Vaccination is widely accepted as the favoured approach to tackle the global healthcare burden of infectious disease and cancer. However, despite significant advances in relation to our understanding of the molecular biology relating to infectious disease and cancers, the development of effective vaccines in these areas has been limited. The most effective vaccines developed have used live, attenuated organisms, however, the safety risk associated with such attenuated pathogens reverting to virulence, has restricted their widespread use. A further major barrier preventing the wide scale development and use of more effective vaccines is the limitation associated with the ability to identify candidate pathogen derived proteins that will elicit broad protective immunity in a specific manner against variant strains of microbial pathogens, when administered as part of a vaccine composition.
One particular approach that shows the promise of conferring broad, protective immunity is the use of stress protein complexes as vaccines against infectious disease and cancer (Colaco et al., (2004) Biochem Soc Trans 32:626-628 and Zeng et al., (2006) Cancer Immunol Immunother 55:329-338)). It has also been widely documented that heat shock protein/antigenic peptide complexes are efficacious as vaccines against specific cancers (U.S. Pat. No. 5,997,873; U.S. Pat. No. 5,935,576, U.S. Pat. No. 5,750,119, U.S. Pat. No. 5,961,979 and U.S. Pat. No. 5,837,251). Colaco and colleagues have shown that pathogen derived stress protein-peptide complexes isolated from heat-shocked BCG cells induced T-helper 1 (Th1) mediated immune responses in a vaccinated subject, which conferred protective immunity against a live challenge in a murine aerosol challenge model of pulmonary tuberculosis (International Patent Application No. WO 01/13944). Moreover, it has been shown in International Patent Application Nos. WO 02/20045, WO 00/10597 and WO 01/13943 that stress protein complexes isolated from pathogens or pathogen infected cells are effective as the immunogenic determinant within vaccines against infectious diseases.
Heat shock proteins (hsps) form a family of highly conserved proteins that are widely distributed throughout the plant and animal kingdoms. On the basis of their molecular weight (kDa), the major heat shock proteins are grouped into six different families: small (hsps of 20-30 kDa); hsp40; hsp60; hsp70; hsp90; and hsp100. Although heat shock proteins were originally identified in cells subjected to heat stress, they have been found to be associated with many other forms of stress, such as infections, osmotic stress, cytokine stress and the like. Accordingly, heat shock proteins are also commonly referred to as stress proteins (SPs) on the basis that their expression is not solely caused by a heat stress. Members of the hsp60 family include the major chaperone GroEL. These form multimeric complexes with co-chaperones such as GroES. Many microbial pathogens have additional hsp60 families that form distinct complexes from GroEL and some hsp60 family members may be more immunogenic, such as the hsp65 of mycobacteria. Members of the hsp70 family include DnaK and these hsp 70 family members also form multimeric complexes with co-chaperones such as DnaJ. Other major heat shock proteins include the AAA ATPases, the Clp proteins, Trigger factor, Hip, HtpG, NAC, Clp, GrpE, SecB and prefoldin.
Stress proteins are ubiquitously expressed in both prokaryotic and eukaryotic cells, where they function as chaperones in the folding and unfolding of polypeptides. A further role of stress proteins is to chaperone peptides from one cellular compartment to another and, in the case of diseased cells, stress proteins are also known to chaperone viral or tumour-associated peptides to the cell-surface. The chaperone function of stress proteins is accomplished through the formation of complexes between stress proteins and the chaperoned polypeptide. Such polypeptides may include peptide fragments.
In the immune response, heterologous polypeptides, or polypeptide fragments complexed with the stress proteins form stress protein-peptide complexes, which may be referred to as heat shock protein complexes (HspCs). HspCs are captured by antigen presenting cells (APCs) to provide a source of antigenic peptides which can be loaded onto major histocompatibility complex (MHC) molecules for cell surface presentation to the T-cells of the immune system.
HspCs have been widely studied as cancer vaccines and methods have been developed for the isolation of HspCs from tumour cells for use as effective vaccines (U.S. Pat. No. 5,997,873; U.S. Pat. No. 5,935,576; U.S. Pat. No. 5,750,119, U.S. Pat. No. 5,961,979 and U.S. Pat. No. 5,837,251). However, these methods resulted in the isolation of restricted families of heat shock proteins, and therefore specifically excluded the use of multiple chaperone proteins as the immunogenic determinant in vaccines. The use of HspCs as cancer vaccines can be significantly improved by the use of multiple chaperone proteins, in particular heat shock proteins (reviewed in Zeng et. al. Cancer Immunol. Immunother (2006) 55: 329-338) and thus methods have been developed for the purification of multiple chaperone proteins and chaperone protein complexes for use in vaccines. For example, U.S. Pat. No. 6,875,849 discloses the use of free-solution isoelectric focusing (FF-IEF) for the purification of HspCs from tumours for use as cancer vaccines.
The present inventors have previously found that FF-IEF can also be used to isolate HspCs from pathogens and infected cells for use as the immunogenic determinant in vaccine compositions for the prevention and treatment of infectious diseases. However, a key limitation of this technique has been the difficulties associated with developing a large scale FF-IEF instrument to produce the quantities of heat shock protein/peptide complexes (HspCs) which would be required for large scale, commercial GMP vaccine manufacture. Additionally, FF-IEF separates complexes on the basis of their isoelectric points (pI) and the process of free-flow focussing is slow with a typical run time of 4 hours, during which high levels of protein degradation result, severely limiting the use of FF-IEF in large scale production of purified heat shock protein-peptide complexes. Furthermore, the use of chaotropes, such as urea, to maintain protein solubility during purification can result in the disruption of some heat shock protein-peptide complexes. Moreover, the use of ampholytes (ampholines) to produce the pH gradient required during the FF-IEF process results in the introduction of a further contaminant, in addition to the chaotropes, in the heat shock protein-peptide containing preparations. Such contaminants, being immunogenic themselves and also unacceptable to Regulatory Authorities, pose a significant barrier to the use of FF-IEF in the manufacture of heat shock protein-peptide complex containing vaccine compositions.